CN107893075A - CRISPR Cas9 targeting knock out people colon-cancer cell RITA genes and its specific sgRNA - Google Patents

Abstract

The invention discloses CRISPR/Cas9 targeting knock out people colon-cancer cell RITA genes and its specific sgRNA.It is the sgRNA for obtaining selectively targeted RITA genes Second Exon first, its base sequence is as shown in SEQ ID NO.1；Next to that the sgRNA of structure RITA genes contains Cas9 albumen to slow virus carrier system, the system；Finally by the cells of CRISPR/Cas9 slow-virus infection people's intestinal cancer HT 29 containing the sgRNA, the significantly reduced cell line of RITA protein expression levels is obtained.The present invention has that operating procedure is simple, sgRNA targetings are good and high to RITA genes cutting efficiency；In addition, constructed CRISPR/Cas9 slow virus system has the advantage for knocking out efficiency high, and energy specific knockdown RITA genes, obtain knocking out people's colon-cancer cell of RITA genes, so as to provide strong instrument further to study the mechanism of action of RITA in colon-cancer cell.

Description

CRISPR-Cas9 targeting knock out people colon-cancer cell RITA genes and its specific sgRNA

Technical field

The invention belongs to genetic engineering field, more specifically to CRISPR/Cas9 targeting knock out people's colon-cancer cells RITA genes and its specific sgRNA.

Background technology

RBP-J is interacted and microtubule associated protein (RITA;C12orf52 it is) a highly conserved albumen, albumen size For 36kD, there is no significant homology with any other protein.RITA can disturb the transcription that Notch and RBP-J is mediated. RITA quickly shuttles between nucleus and cytoplasm, it is most important that, it mediates the core output of RBP-J tubulin fibers. RITA, the RBP-J new as one interact albumen, by disturbing transcription factor RBP-J to lower Notch.Notch gene codes one The highly conserved cell surface receptor of class, they adjust the development of a variety of biological cells such as from sea urchin to people.Notch effect of signals Multiple processes that cell normal morphology occurs, including the specialization of multipotency progenitor cells, Apoptosis, cell propagation and cell boundaries Formed.Transcription factor RBP-J has very important effect in Notch signal paths are regulated and controled, because it is suppressing and activated multiple DNA target sequence is identified in compound.

There are some researches show the expression and downward of the RITA overexpression increase p53 and FBXW7 albumen in liver cancer cells Cyclin D1, cyclin E, CDK2 and NF- κ B p65 expression.These changes cause SMMC-7721 and HepG2 cells Growth inhibition and induction G₀/G₁Phase cell-cycle arrest and apoptosis.It is by thin that the result, which shows that RITA plays tumor inhibition effect, Born of the same parents' cycle G₀/G₁Phase retardance is relevant with inducing cell apoptosis.RITA genes participate in cell cycle regulation, because tumour is likely to be Because caused by cell hyperproliferation, therefore, it is necessary to study mechanism of action of the RITA in different cells.

Intestinal cancer refers to the carninomatosis class disease for betiding enteron aisle, is malignant tumour common in intestines and stomach, the incidence of disease is only second to Stomach cancer and the cancer of the esophagus.At present, researcher has done numerous studies in terms of the mechanism of intestinal cancer, it was found that numerous participation intestinal cancer The approach and mechanism of progress, such as protooncogene activation, tumor suppressor gene inactivate (point mutation, rearrangement, missing) and chromosome abnormality, But current intestinal cancer occurs still have many not yet clearly, it is necessary to further study with molecular mechanism involved in progress.

CRISPR/Cas systems are to rise the important tool for the specific DNA modification of target gene recent years, are had fast The advantage such as fast, efficient and selectively targeted, has been applied to various modes biology, including people, mouse, rat, zebra fish, beautiful Hidden rhabditida, plant and bacterium.CRISPR RNA（crRNA）Can specifically it know with transcriptional activation crRNA compounds to be formed of annealing Other genome sequence, guiding Cas9 endonucleases are in purpose fragment generation DNA double chain fracture.This recognition complex can be with Simplified by merging crRNA and tracrRNA sequences formation sgRNA.There is the fragment for being about 20bp in the target sequence of genome With crRNA or sgRNA complementary pairings；The trinucleotide region PAM of target sequence end（5’-NGG-3’）Position is identified for spCas9 Point, it is the key for realizing shearing function.Can sgRNA accomplish that specificity, accurate targeting target gene are CRISPR/Cas9 spies The opposite sex knocks out the prerequisite of target gene, misses the target and mistake targeting can all influence CRISPR/Cas9 to the special of target gene Property knock out, turn into CRISPR/Cas9 therefore, it is possible to design, prepare the sgRNA of accuracy and selectively targeted target gene The key technology of gene knockout.

The present invention utilizes CRISPR/Cas9 slow virus systems, obtains the people's colon-cancer cell for knocking out RITA genes, to study intestines RITA mechanism of action provides powerful in cancer cell.

Bibliography, 1）Horvath P, Barrangou R (January 2010). "CRISPR/Cas, the immune system of bacteria and archaea". Science 327 (5962): 167–170；2）Hale CR, Zhao P, Olson S, et al. (November 2009). "RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex". Cell139 (5): 945–956；3）Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A., and Charpentie E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptiv bacterial immunity. Science337, 816–821；4）Hsu, P.D., Scott, D.A.,Weinstein, J.A., Ran, F.A., Konermann, S., Agarwala, V.,Li, Y., Fine, E.J., Wu, X., Shalem, O., et Al. (2013) DNA targeting speci city of RNA-guided Cas9 nucleases.Nat. Biotechnol. Published online July 21, 2013；5）Haihe Wang, Zhanchun Yang , Chunbo Liu , Shishun Huang , Hongzhi Wang, Yingli Chen ,and Guofu Chen. (October 2014). RBP-J-interacting and tubulin-associated protein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma by activating the p53–Fbxw7 pathway. Biochemical and Biophysical Research Communications 454 (2014) 71–77；6）S.A. Wacker, C. Alvarado, G. von Wichert, et al., RITA, a novel modulator of Notch signalling, acts via nuclear export of RBP-J, EMBO J. 30 (2011) 43–56。

The content of the invention

The shortcomings that primary and foremost purpose of the present invention is to overcome prior art and deficiency, there is provided a kind of targeting knock out RITA genes SgRNA sequences.

Another object of the present invention is to provide a kind of CRISPR/Cas9 slow virus system of targeting knock out RITA genes The application of system.

It is still another object of the present invention to provide the application of people's colon-cancer cell HT-29 cells of targeting knock out RITA genes.

The purpose of the present invention is achieved through the following technical solutions：A kind of sgRNA sequences of targeting knock out RITA genes, are selected from The following RITA sgRNA of DNA sequence dna：

RITA sgRNA sequence is as follows：

RITA sgRNA oligo1：5’-CACCGCACCGGACTTCGATCCGCCC-3’；

RITAsgRNA oligo2：5’-AAACGGGCGGATCGAAGTCCGGTGC-3’；

A kind of CRISPR/Cas9 slow virus systems of targeting knock out RITA genes, contain above-mentioned targeting knock out RITA genes SgRNA DNA sequence dna.

The structure of the CRISPR/Cas9 slow virus systems of described targeting knock out RITA genes, comprises the following steps：

(1) BsmBI digestion CRISPR/Cas9 slow virus carrier Lenti-CRISPRv2 are used, after obtaining digestion CRISPR/

Cas9 slow virus carriers；

(2) by after the sgRNA of above-mentioned targeting knock out RITA genes DNA sequence dna phosphorylation with the CRISPR/Cas9 after digestion

Slow virus carrier connects, and obtains the CRISPR/Cas9 slow virus systems of targeting knock out RITA genes；

DNA sequence dna described in step (2) is by oligonucleotide chain 1 (oligo1) and oligonucleotide chain 2 (oligo2) Annealing obtains double-stranded sequence；

The CRISPR/Cas9 slow virus system of described targeting knock out RITA genes is in the cell line for knocking out RITA genes is prepared Application；

A kind of cell line of knockout RITA genes, it is by the CRISPR/Cas9 slow virus system of described targeting knock out RITA genes System transfection aim cell strain obtains.

The cell line of described knockout RITA genes, builds to obtain particular by following steps：

1) the CRISPR/Cas9 slow virus system of described targeting knock out RITA genes is packed by incasing cells, obtained To lentiviral particle；

2) lentiviral particle is infected into aim cell strain；

3）Metainfective cell is spread into monoclonal, obtains knocking out the cell line of RITA genes.

Described aim cell strain is preferably tumor cell line.

Described tumor cell line is preferably colon-cancer cell strain.

Described colon-cancer cell strain is preferably human colon carcinoma immortalized cells HT-29.

The cell line of described knockout RITA genes, it is that RITA gene delections in HT-29 cells or insertion nucleotides obtain The cell line arrived, it is any strain in following cell line：

Cell line 3# is：SgRNA cleavage site at 7 bases before sgRNA PAM areas on wild type RITA genes, The second exon of RITA genes is caused to lack 134 bases；Cell line 5#：Be sgRNA cleavage site be located at wild Before sgRNA PAM areas on type RITA genes at 33 bases, the second exon of RITA genes is caused to lack 160 alkali Base.；The specific targeting knock out site of RITA genes is with wild type RITA gene orders to such as Fig. 6 and Fig. 7.

The present invention is had the following advantages relative to prior art and effect：

The present invention provides the sgRNA energy special target RITA genes of RITA genes, is built into CRISPR/Cas9 slow virus system System, the system energy special target knock out RITA genes, obtain knocking out the cell line of RITA genes, so as to be advantageous to study cell line In RITA mechanism of action.

Brief description of the drawings

Fig. 1 is the vector plasmid Lenti-CRISPR v2 used in the embodiment of the present invention plasmid map；

Fig. 2 is the expression vector plasmid Lenti-CRISPRv2-hRITAsg used in the embodiment of the present invention plasmid map；

Fig. 3 is the packaging plasmid pLP-VSVG used in the embodiment of the present invention plasmid map；

Fig. 4 is the packaging plasmid psPAX2 used in the embodiment of the present invention plasmid map；

Fig. 5 is that HT-29 cell virus infects the activity identification sequencer map containing RITAsgRNA；

Fig. 6 is the sequencer map of RITA genes in the monoclonal HT-29 cell lines 3 for knock out RITA genes；

Fig. 7 is the sequencer map of RITA genes in the monoclonal HT-29 cell lines 5 for knock out RITA genes.

Embodiment

With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited In this.

Embodiment 1

1, knocks out RITA plasmids using CRISPR/Cas9 technique constructions

1 .1sgRNA oligonucleotide chains synthesize

Use CRISPR Photographing On-line instruments (http://crispr.mit.edu/) according to points-scoring system, the second of RITA 1 20bp sgRNA is designed on extron, and is verified by BLAST without non-specific gene.The addition of the end of coding strand template 5 ' The cohesive end formed after ACCG, the addition of the end of noncoding strand template 3 ' AAAC, with BsmBI digestions is complementary, designs 1 couple of CRISPR widow Nucleotide chain, is shown in Table 1, the RITA target sites of table 1 and sgRNA oligonucleotide sequences.

1 .2 vector constructions

1.2.1 the μ g Lenti-CRISPRv2 plasmids (being purchased from Addgene companies) of BsmBI digestions 2 are used, 2h, 37 DEG C,

Digestion system is：

1.2.2 JaRa gene glue reclaim kits digested plasmid product is used, by specification is operated.

1.2.3 phosphorylation and the sgRNA oligos that anneal, enzyme disjunctor system are：

PCR instrument device cycle of annealing：37 DEG C of 30min, 95 DEG C of maintenance 5min, per minute to reduce by 5 DEG C to 25 DEG C, 4 DEG C of maintenances.

1.2.4 the Lenti-CRISPRv2 carriers after anneal the Oligo double-strands formed and digestion are directly connected to, room temperature Under, 10min.

1.2.5 the plasmid after connection is converted into competent cell DH5 α, is uniformly applied in LB solid medium flat boards, Culture 12-16 hours in 37 DEG C of incubators are placed in, single bacterium colony may occur in which.

The expansion of 1 .3 pickings single bacterium colony, which is cultivated, and plasmid is small carries.

The success of 1.4 sequencing identification plasmid constructions, and it is named as Lenti-CRISPRv2-hRITAsg.

1.5 knock out efficiency checking, and the DMEM in high glucose culture containing 10% hyclone is based on 5%CO2, and 37 DEG C incubated 293T cells (are purchased from U.S.'s ATCC cell banks).Phase cell take the logarithm with 1 × 10⁵/ hole is inoculated into 24 orifice plate cultures.Treat that cell melts It is right to be replaced with Opti-MEM culture mediums when reaching 70%~8 0 %, corresponding CRISPR/Cas9 is knocked out into matter after 1 hour 0.8 μ g of grain are transfected into 293T cells, as a child, the transfection of fluorescence microscopy Microscopic observation is imitated for transfection 48 through Lipo2000 reagents Cell is collected in fruit, digestion, and cell genomic dna is extracted using genome DNA extracting reagent kit；With each group cell genomic dna For template, the primer of table 2 is utilized（Seq No.4~7）PCR amplification targeting sequences.Carried out with RITA-4-1 and RITA-4-2 PCR, 930bp fragment a is obtained, using a as template, enters performing PCR with RITA-4-3 and RITA-4-4, obtain 404bp fragment b, Glue reclaim is used to be sequenced after the fragment electrophoretic, sequencing primer RITA-4-3, the primer situation table of table 2.

2, pack slow virus

100mm ware kinds enter 4X10⁶Individual 293TN cells, after cultivating 24h (37 DEG C, 5%CO2), fresh culture is changed, is turned Contaminate incasing cells：

A mix following 3 plasmids：1）The μ g of packaging plasmid psPAX2 12；The μ g of helper plasmid pLP-VSVG 10；Expression plasmid Lenti-CRISPRv2- hRITAsg 22μg；Add CaCl₂250μl；Add ddH₂O is 500 μ l to cumulative volume, and 37 DEG C are placed 20min；

B .2 X BES 500 μ l, 37 DEG C of placement 20min；

C the plasmid mixed liquors of 500 μ l in a tri- are added dropwise in b, and are gently mixed；

D. it is careful that mixed liquor addition kind in c has in 293TN 100mm wares；Infection liquid is removed after cultivating 12h, changes 10ml Fresh culture produces virus liquid；After cultivating 48h, harvest virus liquid is mended into 100mm wares again into 50ml centrifuge tubes 10ml culture mediums are added to produce virus again；After cultivating 24h, virus liquid, room temperature centrifugation, 1000rpm, 10min are harvested again；Use 0.22 μm of filter filter virus supernatant；Vial supernatant is fitted into and surpassed from pipe, 4 DEG C, 100000g, 2h；Abandoning supernatant It is resuspended overnight with Opti-MEM afterwards；Reuse the virus that 0.22 μm of filter filtering is resuspended；After packing, -80 DEG C of preservations.

3, virus infection

1) the HT-29 cells (being purchased from U.S.'s ATCC cell banks) that will be infected are inoculated into six orifice plates, overnight, treat cell fusion During degree about 50%, culture medium is removed, changes 2ml fresh cultures；

2) 20 μ l virus liquids are added per hole, add 12 μ l polybrene polybrene, to final concentration of 6 μ g/ml；

3) after 37 DEG C of culture 12h, nutrient solution is removed, continues to cultivate 48h after changing fresh medium.

4, screen stable cell line

After infection terminates 48h, puromycin (2 .0 μ g/ml) is added to every hole, changes liquid every other day, and keep the purine of culture medium Mycin constant concentration, screening positive clone cell.Gained cell line is named as HT-29-RITA.And selected using limiting dilution assay HT-29-RITA monoclonals knock out cell line.

5, stable cell lines are identified and gene targeting mode is identified

The monoclonal cell strain genomic DNA for knocking out RITA using each group is separately designed for RITA Second Exons as template Primer, sequence such as Seq No.4~7 carry out two-wheeled nested PCR amplification, the second wheel PCR products of acquisition are sequenced, such as There is bimodal situation in sequencing result target practice location proximate, then may be to practice shooting successfully.Bimodal sample PCR again is selected, is produced TA is cloned into T carriers after thing glue reclaim, and picking positive colony is sequenced again after conversion, such as sequencing result RITA bases Because target site nearby occur non-triple base insertion or base deletion, cause frame shift mutation, then can determine whether for RITA gene knockouts.It is active to identify sgRNA（Fig. 5）, and find 2 types have RITA gene delections or The stable cell line (Fig. 6 and Fig. 7) of insertion mutation.

Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Sequence table

<110>With first biotechnology (Shanghai) limited company

<120>CRISPR-Cas9 targeting knock out people colon-cancer cell RITA genes and its specific sgRNA

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 1

aaccggactt cgatccgccc 20

<210> 2

<211> 25

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 2

caccgcaccg gacttcgatc cgccc 25

<210> 3

<211> 25

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 3

aaacgggcgg atcgaagtcc ggtgc 25

<210> 4

<211> 19

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 4

tgtcatctct ccttcaccg 19

<210> 5

<211> 18

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 5

gactcctctg ggcctcag 18

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<211> 29

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

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cccaagcttc tctgacctcc tctcacagg 29

<210> 7

<211> 28

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 7

ccggaattcc atctccctac cccattac 28

<210> 8

<211> 14873

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 8

tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 60

caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 120

ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 180

gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc 240

tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 300

ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 360

ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga 420

aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca 480

cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct 540

agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat 600

gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat 660

gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag 720

gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta 780

gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag 840

acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac 900

agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt 960

gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca 1020

aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg 1080

ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc 1140

agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg 1200

caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg 1260

gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa 1320

ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag 1380

atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta 1440

atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg 1500

gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat 1560

ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta 1620

ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc 1680

ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 1740

agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca 1800

aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 1860

ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 1920

tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat 1980

taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 2040

gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg 2100

tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 2160

gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 2220

tggaaaggac gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa 2280

tattatattc tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat 2340

caataattag aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa 2400

aaaacgctag ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc 2460

acttgttcct aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct 2520

aactccacca gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac 2580

atcttcaaat gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc 2640

agcttcaaca gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc 2700

cataatacat ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa 2760

aactaattca taatttttgt cttttaatgc accaatattt aataccatat caatttctgt 2820

tgcaccatct ttaattgctt cagaaacttc gaatgctttt gtagctgttg tgcatgcacc 2880

tagaggaaaa cctacaacat ttgttattcc tacatttgtg ccttttaata attctttaca 2940

atagcttgtt caatatgaat taacacaaac tgttgcaaaa tcaaattcaa ttgcttcatc 3000

acataattgt ttaatttcag ctttcgtagc atcttgtttt aataatgtgt gatctatata 3060

tttgtttagt ttcatttttt ctcctatata ttcattttta attttaattc tttaataatt 3120

tcgtctactt taactttagc gttttgaaca gattcaccaa cacctataaa ataaattttt 3180

agtttaggtt cagttccact tgggcgaaca gcaaatcatg acttatcttc taaataaaat 3240

tttagtaagt cttgtcctgg catattatac attccatcga tgtagtcttc aacattaaca 3300

actttaagtc cagcaatttg agttaagggt gttgctctca atgatttcat taatggttca 3360

atttttaatt tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca 3420

cccatttctt taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa 3480

aatcaagcag cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag 3540

tcatcaatta catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct 3600

tcttctttag caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta 3660

actccatatt tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat 3720

agagccggat tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt 3780

aaaattggtc ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc 3840

attccaaatc tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata 3900

ccatattcaa gcggtatttt tcatgcagga tcaaattctg gatttggatt tacaacattt 3960

ttaaatgttt catcttcaaa tgcatgctct tcaacctcaa taacgttata tcctgattca 4020

cgtaatattt ttggggtaaa tttagttcct gttccattaa ctgcgctaaa aataattttt 4080

aaatcttttt tagcttcttg ctcttttttg tacgtctctg ttttagagct agaaatagca 4140

agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 4200

tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc 4260

agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg 4320

tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct 4380

ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt 4440

tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa 4500

gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga 4560

cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 4620

caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 4680

gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 4740

agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 4800

gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 4860

ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 4920

ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 4980

gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 5040

gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 5100

cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 5160

aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc 5220

cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa 5280

actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 5340

cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 5400

cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 5460

gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 5520

gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 5580

tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 5640

ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 5700

gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct 5760

gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 5820

cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 5880

ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga 5940

caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 6000

caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 6060

gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 6120

gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 6180

gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 6240

ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 6300

caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 6360

gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 6420

cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 6480

ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 6540

gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac 6600

ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 6660

cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 6720

ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 6780

ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 6840

gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 6900

cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 6960

cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc 7020

tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 7080

ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 7140

gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 7200

cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 7260

aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 7320

cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 7380

gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 7440

cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 7500

gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 7560

caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 7620

gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 7680

cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 7740

gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 7800

cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 7860

gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 7920

gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 7980

gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 8040

agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 8100

gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg 8160

aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 8220

gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 8280

gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 8340

cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 8400

agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 8460

cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 8520

ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 8580

tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa 8640

gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa 8700

acaagccgga gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct 8760

cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta 8820

ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca 8880

agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg 8940

cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga 9000

gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga 9060

aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt 9120

ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc 9180

ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt 9240

ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac 9300

ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga 9360

ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 9420

tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 9480

ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 9540

gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 9600

caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 9660

actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 9720

ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 9780

ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 9840

tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 9900

gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac 9960

aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt 10020

cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag 10080

atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 10140

ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 10200

tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg 10260

atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 10320

ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 10380

atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 10440

gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 10500

tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc 10560

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 10620

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 10680

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 10740

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 10800

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 10860

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 10920

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg 10980

aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 11040

agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 11100

agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 11160

cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 11220

ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga 11280

ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt 11340

ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat 11400

aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca 11460

ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg 11520

acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg 11580

cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg 11640

acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc 11700

cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg 11760

ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg 11820

attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 11880

ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta 11940

ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 12000

ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 12060

gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 12120

gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 12180

cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 12240

tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 12300

gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 12360

ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 12420

caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 12480

aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 12540

atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 12600

cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 12660

ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 12720

gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 12780

accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 12840

cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 12900

cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 12960

gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 13020

aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 13080

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 13140

actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 13200

taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 13260

gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 13320

tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 13380

ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 13440

accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 13500

agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 13560

acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 13620

tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 13680

cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 13740

tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 13800

ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 13860

gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 13920

tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 13980

ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 14040

gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 14100

cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 14160

gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 14220

ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga 14280

tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct 14340

gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac 14400

aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct 14460

gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata 14520

gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 14580

tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 14640

gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta 14700

tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 14760

tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 14820

ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgg 14873

<210> 9

<211> 13012

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 9

tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 60

caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 120

ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 180

gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc 240

tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 300

ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 360

ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga 420

aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca 480

cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct 540

agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat 600

gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat 660

gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag 720

gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta 780

gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag 840

acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac 900

agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt 960

gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca 1020

aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg 1080

ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc 1140

agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg 1200

caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg 1260

gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa 1320

ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag 1380

atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta 1440

atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg 1500

gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat 1560

ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta 1620

ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc 1680

ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 1740

agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca 1800

aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 1860

ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 1920

tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat 1980

taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 2040

gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg 2100

tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 2160

gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 2220

tggaaaggac gaaacaccca ccggacttcg atccgcccgt tttagagcta gaaatagcaa 2280

gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 2340

gaattcgcta gctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca 2400

gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgatccggt 2460

gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt 2520

tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt 2580

cgcaacgggt ttgccgccag aacacaggac cggttctaga gcgctgccac catggacaag 2640

aagtacagca tcggcctgga catcggcacc aactctgtgg gctgggccgt gatcaccgac 2700

gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg gcacagcatc 2760

aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga ggccacccgg 2820

ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg ctatctgcaa 2880

gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag actggaagag 2940

tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg caacatcgtg 3000

gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa gaaactggtg 3060

gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca catgatcaag 3120

ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga cgtggacaag 3180

ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc catcaacgcc 3240

agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag acggctggaa 3300

aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa cctgattgcc 3360

ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga ggatgccaaa 3420

ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc ccagatcggc 3480

gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat cctgctgagc 3540

gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc tatgatcaag 3600

agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg 3660

cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc cggctacatt 3720

gacggcggag ccagccagga agagttctac aagttcatca agcccatcct ggaaaagatg 3780

gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg gaagcagcgg 3840

accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca cgccattctg 3900

cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat cgagaagatc 3960

ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag cagattcgcc 4020

tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga agtggtggac 4080

aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa gaacctgccc 4140

aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt gtataacgag 4200

ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct gagcggcgag 4260

cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag 4320

ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg 4380

gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat tatcaaggac 4440

aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt gctgaccctg 4500

acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc ccacctgttc 4560

gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg caggctgagc 4620

cggaagctga tcaacggcat ccgggacaag cagtccggca agacaatcct ggatttcctg 4680

aagtccgacg gcttcgccaa cagaaacttc atgcagctga tccacgacga cagcctgacc 4740

tttaaagagg acatccagaa agcccaggtg tccggccagg gcgatagcct gcacgagcac 4800

attgccaatc tggccggcag ccccgccatt aagaagggca tcctgcagac agtgaaggtg 4860

gtggacgagc tcgtgaaagt gatgggccgg cacaagcccg agaacatcgt gatcgaaatg 4920

gccagagaga accagaccac ccagaaggga cagaagaaca gccgcgagag aatgaagcgg 4980

atcgaagagg gcatcaaaga gctgggcagc cagatcctga aagaacaccc cgtggaaaac 5040

acccagctgc agaacgagaa gctgtacctg tactacctgc agaatgggcg ggatatgtac 5100

gtggaccagg aactggacat caaccggctg tccgactacg atgtggacca tatcgtgcct 5160

cagagctttc tgaaggacga ctccatcgac aacaaggtgc tgaccagaag cgacaagaac 5220

cggggcaaga gcgacaacgt gccctccgaa gaggtcgtga agaagatgaa gaactactgg 5280

cggcagctgc tgaacgccaa gctgattacc cagagaaagt tcgacaatct gaccaaggcc 5340

gagagaggcg gcctgagcga actggataag gccggcttca tcaagagaca gctggtggaa 5400

acccggcaga tcacaaagca cgtggcacag atcctggact cccggatgaa cactaagtac 5460

gacgagaatg acaagctgat ccgggaagtg aaagtgatca ccctgaagtc caagctggtg 5520

tccgatttcc ggaaggattt ccagttttac aaagtgcgcg agatcaacaa ctaccaccac 5580

gcccacgacg cctacctgaa cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag 5640

ctggaaagcg agttcgtgta cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc 5700

aagagcgagc aggaaatcgg caaggctacc gccaagtact tcttctacag caacatcatg 5760

aactttttca agaccgagat taccctggcc aacggcgaga tccggaagcg gcctctgatc 5820

gagacaaacg gcgaaaccgg ggagatcgtg tgggataagg gccgggattt tgccaccgtg 5880

cggaaagtgc tgagcatgcc ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc 5940

ggcttcagca aagagtctat cctgcccaag aggaacagcg ataagctgat cgccagaaag 6000

aaggactggg accctaagaa gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg 6060

ctggtggtgg ccaaagtgga aaagggcaag tccaagaaac tgaagagtgt gaaagagctg 6120

ctggggatca ccatcatgga aagaagcagc ttcgagaaga atcccatcga ctttctggaa 6180

gccaagggct acaaagaagt gaaaaaggac ctgatcatca agctgcctaa gtactccctg 6240

ttcgagctgg aaaacggccg gaagagaatg ctggcctctg ccggcgaact gcagaaggga 6300

aacgaactgg ccctgccctc caaatatgtg aacttcctgt acctggccag ccactatgag 6360

aagctgaagg gctcccccga ggataatgag cagaaacagc tgtttgtgga acagcacaag 6420

cactacctgg acgagatcat cgagcagatc agcgagttct ccaagagagt gatcctggcc 6480

gacgctaatc tggacaaagt gctgtccgcc tacaacaagc accgggataa gcccatcaga 6540

gagcaggccg agaatatcat ccacctgttt accctgacca atctgggagc ccctgccgcc 6600

ttcaagtact ttgacaccac catcgaccgg aagaggtaca ccagcaccaa agaggtgctg 6660

gacgccaccc tgatccacca gagcatcacc ggcctgtacg agacacggat cgacctgtct 6720

cagctgggag gcgacaagcg acctgccgcc acaaagaagg ctggacaggc taagaagaag 6780

aaagattaca aagacgatga cgataaggga tccggcgcaa caaacttctc tctgctgaaa 6840

caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac ggtgcgcctc 6900

gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac 6960

cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa 7020

gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc 7080

gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag 7140

atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa 7200

ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc 7260

tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg 7320

gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc 7380

tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc 7440

tggtgcatga cccgcaagcc cggtgcctga acgcgttaag tcgacaatca acctctggat 7500

tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt 7560

ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc 7620

tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg 7680

caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc 7740

accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa 7800

ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 7860

tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc 7920

tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt 7980

ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag 8040

acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac tttaagacca atgacttaca 8100

aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc 8160

actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga 8220

tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct 8280

tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat 8340

ccctcagacc cttttagtca gtgtggaaaa tctctagcag ggcccgttta aacccgctga 8400

tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct 8460

tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca 8520

tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag 8580

ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct 8640

gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca 8700

ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 8760

gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 8820

caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 8880

cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 8940

tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 9000

acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 9060

gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga 9120

atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 9180

gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca 9240

gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc 9300

ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt 9360

tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag 9420

gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt 9480

tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg gcatagtata 9540

atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc cggtgctcac 9600

cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga 9660

cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc 9720

ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga 9780

cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc 9840

ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc 9900

cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac gagatttcga 9960

ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg 10020

gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat 10080

tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 10140

tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg 10200

tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg 10260

aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 10320

ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 10380

ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 10440

cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 10500

tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 10560

aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 10620

aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 10680

tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 10740

ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 10800

cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 10860

ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 10920

ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 10980

gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 11040

agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 11100

cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 11160

aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 11220

aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 11280

ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 11340

aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 11400

ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 11460

agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 11520

cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 11580

ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 11640

gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 11700

cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 11760

cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 11820

ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 11880

catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 11940

tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 12000

ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 12060

catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 12120

cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 12180

cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 12240

acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 12300

ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 12360

tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat 12420

cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg 12480

ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca 12540

aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg 12600

cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag 12660

taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt 12720

acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg 12780

acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat 12840

ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct 12900

attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg 12960

gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gg 13012

<210> 10

<211> 6507

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 10

gcggccgctc tagagagctt ggcccattgc atacgttgta tccatatcat aatatgtaca 60

tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg actagttatt 120

aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 180

aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 240

taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 300

agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 360

cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 420

tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 480

tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 540

gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 600

caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 660

aggtctatat aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac 720

gctgttttga cctccataga agacaccggg accgatccag cctccggtcg accgatcctg 780

agaacttcag ggtgagtttg gggacccttg attgttcttt ctttttcgct attgtaaaat 840

tcatgttata tggagggggc aaagttttca gggtgttgtt tagaatggga agatgtccct 900

tgtatcacca tggaccctca tgataatttt gtttctttca ctttctactc tgttgacaac 960

cattgtctcc tcttattttc ttttcatttt ctgtaacttt ttcgttaaac tttagcttgc 1020

atttgtaacg aatttttaaa ttcacttttg tttatttgtc agattgtaag tactttctct 1080

aatcactttt ttttcaaggc aatcagggta tattatattg tacttcagca cagttttaga 1140

gaacaattgt tataattaaa tgataaggta gaatatttct gcatataaat tctggctggc 1200

gtggaaatat tcttattggt agaaacaact acaccctggt catcatcctg cctttctctt 1260

tatggttaca atgatataca ctgtttgaga tgaggataaa atactctgag tccaaaccgg 1320

gcccctctgc taaccatgtt catgccttct tctctttcct acagctcctg ggcaacgtgc 1380

tggttgttgt gctgtctcat cattttggca aagaattcct cgacggatcc ctcgaggaat 1440

tctgacacta tgaagtgcct tttgtactta gcctttttat tcattggggt gaattgcaag 1500

ttcaccatag tttttccaca caaccaaaaa ggaaactgga aaaatgttcc ttctaattac 1560

cattattgcc cgtcaagctc agatttaaat tggcataatg acttaatagg cacagcctta 1620

caagtcaaaa tgcccaagag tcacaaggct attcaagcag acggttggat gtgtcatgct 1680

tccaaatggg tcactacttg tgatttccgc tggtatggac cgaagtatat aacacattcc 1740

atccgatcct tcactccatc tgtagaacaa tgcaaggaaa gcattgaaca aacgaaacaa 1800

ggaacttggc tgaatccagg cttccctcct caaagttgtg gatatgcaac tgtgacggat 1860

gccgaagcag tgattgtcca ggtgactcct caccatgtgc tggttgatga atacacagga 1920

gaatgggttg attcacagtt catcaacgga aaatgcagca attacatatg ccccactgtc 1980

cataactcta caacctggca ttctgactat aaggtcaaag ggctatgtga ttctaacctc 2040

atttccatgg acatcacctt cttctcagag gacggagagc tatcatccct gggaaaggag 2100

ggcacagggt tcagaagtaa ctactttgct tatgaaactg gaggcaaggc ctgcaaaatg 2160

caatactgca agcattgggg agtcagactc ccatcaggtg tctggttcga gatggctgat 2220

aaggatctct ttgctgcagc cagattccct gaatgcccag aagggtcaag tatctctgct 2280

ccatctcaga cctcagtgga tgtaagtcta attcaggacg ttgagaggat cttggattat 2340

tccctctgcc aagaaacctg gagcaaaatc agagcgggtc ttccaatctc tccagtggat 2400

ctcagctatc ttgctcctaa aaacccagga accggtcctg ctttcaccat aatcaatggt 2460

accctaaaat actttgagac cagatacatc agagtcgata ttgctgctcc aatcctctca 2520

agaatggtcg gaatgatcag tggaactacc acagaaaggg aactgtggga tgactgggca 2580

ccatatgaag acgtggaaat tggacccaat ggagttctga ggaccagttc aggatataag 2640

tttcctttat acatgattgg acatggtatg ttggactccg atcttcatct tagctcaaag 2700

gctcaggtgt tcgaacatcc tcacattcaa gacgctgctt cgcaacttcc tgatgatgag 2760

agtttatttt ttggtgatac tgggctatcc aaaaatccaa tcgagcttgt agaaggttgg 2820

ttcagtagtt ggaaaagctc tattgcctct tttttcttta tcatagggtt aatcattgga 2880

ctattcttgg ttctccgagt tggtatccat ctttgcatta aattaaagca caccaagaaa 2940

agacagattt atacagacat agagatgaac cgacttggaa agtaactcaa atcctgcaca 3000

acagattctt catgtttgga ccaaatcaac ttgtgatacc atgctcaaag aggcctcaat 3060

tatatttgag tttttaattt ttatgaaaaa aaaaaaaaaa aacggaattc ctcgagggat 3120

ccgtcgagga attcactcct caggtgcagg ctgcctatca gaaggtggtg gctggtgtgg 3180

ccaatgccct ggctcacaaa taccactgag atctttttcc ctctgccaaa aattatgggg 3240

acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg 3300

caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc 3360

atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgctgg 3420

ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag ccccctgctg 3480

tccattcctt attccataga aaagccttga cttgaggtta gatttttttt atattttgtt 3540

ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta ctagccagat 3600

ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctctta tggagatccc 3660

tcgacggatc ggccgcaatt cgtaatcatg tcatagctgt ttcctgtgtg aaattgttat 3720

ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 3780

taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 3840

aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 3900

attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 3960

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4020

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4080

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4140

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4200

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4260

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 4320

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4380

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 4440

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 4500

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 4560

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 4620

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 4680

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 4740

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 4800

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 4860

ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 4920

ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 4980

atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 5040

ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 5100

tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 5160

attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 5220

tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 5280

ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 5340

gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 5400

gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 5460

gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 5520

aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 5580

taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 5640

tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 5700

tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 5760

atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 5820

tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 5880

atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 5940

aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 6000

tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 6060

cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 6120

atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 6180

cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 6240

tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc 6300

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 6360

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 6420

tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat 6480

agggcgaatt ggagctccac cgcggtg 6507

<210> 11

<211> 10703

<212> DNA

<213>Artificial synthesized (Artificial synthesis)

<400> 11

gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc 420

atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca 480

gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg 540

gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt 600

tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc 660

gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc 720

ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 780

gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 840

ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg 900

tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg 960

cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc 1020

ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 1080

tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc 1140

cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc 1200

gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg 1260

ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct 1320

gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg 1380

gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440

tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt 1500

cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg 1560

acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1620

gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca 1680

acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcgggccg gccgcgttga 1740

cgcgcacggc aagaggcgag gggcggcgac tggtgagaga tgggtgcgag agcgtcagta 1800

ttaagcgggg gagaattaga tcgatgggaa aaaattcggt taaggccagg gggaaagaaa 1860

aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat 1920

cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc 1980

cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt 2040

gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag 2100

caaaacaaaa gtaagaaaaa agcacagcaa gcagcagctg acacaggaca cagcaatcag 2160

gtcagccaaa attaccctat agtgcagaac atccaggggc aaatggtaca tcaggccata 2220

tcacctagaa ctttaaatgc atgggtaaaa gtagtagaag agaaggcttt cagcccagaa 2280

gtgataccca tgttttcagc attatcagaa ggagccaccc cacaagattt aaacaccatg 2340

ctaaacacag tggggggaca tcaagcagcc atgcaaatgt taaaagagac catcaatgag 2400

gaagctgcag aatgggatag agtgcatcca gtgcatgcag ggcctattgc accaggccag 2460

atgagagaac caaggggaag tgacatagca ggaactacta gtacccttca ggaacaaata 2520

ggatggatga cacataatcc acctatccca gtaggagaaa tctataaaag atggataatc 2580

ctgggattaa ataaaatagt aagaatgtat agccctacca gcattctgga cataagacaa 2640

ggaccaaagg aaccctttag agactatgta gaccgattct ataaaactct aagagccgag 2700

caagcttcac aagaggtaaa aaattggatg acagaaacct tgttggtcca aaatgcgaac 2760

ccagattgta agactatttt aaaagcattg ggaccaggag cgacactaga agaaatgatg 2820

acagcatgtc agggagtggg gggacccggc cataaagcaa gagttttggc tgaagcaatg 2880

agccaagtaa caaatccagc taccataatg atacagaaag gcaattttag gaaccaaaga 2940

aagactgtta agtgtttcaa ttgtggcaaa gaagggcaca tagccaaaaa ttgcagggcc 3000

cctaggaaaa agggctgttg gaaatgtgga aaggaaggac accaaatgaa agattgtact 3060

gagagacagg ctaatttttt agggaagatc tggccttccc acaagggaag gccagggaat 3120

tttcttcaga gcagaccaga gccaacagcc ccaccagaag agagcttcag gtttggggaa 3180

gagacaacaa ctccctctca gaagcaggag ccgatagaca aggaactgta tcctttagct 3240

tccctcagat cactctttgg cagcgacccc tcgtcacaat aaagataggg gggcaattaa 3300

aggaagctct attagataca ggagcagatg atacagtatt agaagaaatg aatttgccag 3360

gaagatggaa accaaaaatg atagggggaa ttggaggttt tatcaaagta ggacagtatg 3420

atcagatact catagaaatc tgcggacata aagctatagg tacagtatta gtaggaccta 3480

cacctgtcaa cataattgga agaaatctgt tgactcagat tggctgcact ttaaattttc 3540

ccattagtcc tattgagact gtaccagtaa aattaaagcc aggaatggat ggcccaaaag 3600

ttaaacaatg gccattgaca gaagaaaaaa taaaagcatt agtagaaatt tgtacagaaa 3660

tggaaaagga aggaaaaatt tcaaaaattg ggcctgaaaa tccatacaat actccagtat 3720

ttgccataaa gaaaaaagac agtactaaat ggagaaaatt agtagatttc agagaactta 3780

ataagagaac tcaagatttc tgggaagttc aattaggaat accacatcct gcagggttaa 3840

aacagaaaaa atcagtaaca gtactggatg tgggcgatgc atatttttca gttcccttag 3900

ataaagactt caggaagtat actgcattta ccatacctag tataaacaat gagacaccag 3960

ggattagata tcagtacaat gtgcttccac agggatggaa aggatcacca gcaatattcc 4020

agtgtagcat gacaaaaatc ttagagcctt ttagaaaaca aaatccagac atagtcatct 4080

atcaatacat ggatgatttg tatgtaggat ctgacttaga aatagggcag catagaacaa 4140

aaatagagga actgagacaa catctgttga ggtggggatt taccacacca gacaaaaaac 4200

atcagaaaga acctccattc ctttggatgg gttatgaact ccatcctgat aaatggacag 4260

tacagcctat agtgctgcca gaaaaggaca gctggactgt caatgacata cagaaattag 4320

tgggaaaatt gaattgggca agtcagattt atgcagggat taaagtaagg caattatgta 4380

aacttcttag gggaaccaaa gcactaacag aagtagtacc actaacagaa gaagcagagc 4440

tagaactggc agaaaacagg gagattctaa aagaaccggt acatggagtg tattatgacc 4500

catcaaaaga cttaatagca gaaatacaga agcaggggca aggccaatgg acatatcaaa 4560

tttatcaaga gccatttaaa aatctgaaaa caggaaaata tgcaagaatg aagggtgccc 4620

acactaatga tgtgaaacaa ttaacagagg cagtacaaaa aatagccaca gaaagcatag 4680

taatatgggg aaagactcct aaatttaaat tacccataca aaaggaaaca tgggaagcat 4740

ggtggacaga gtattggcaa gccacctgga ttcctgagtg ggagtttgtc aatacccctc 4800

ccttagtgaa gttatggtac cagttagaga aagaacccat aataggagca gaaactttct 4860

atgtagatgg ggcagccaat agggaaacta aattaggaaa agcaggatat gtaactgaca 4920

gaggaagaca aaaagttgtc cccctaacgg acacaacaaa tcagaagact gagttacaag 4980

caattcatct agctttgcag gattcgggat tagaagtaaa catagtgaca gactcacaat 5040

atgcattggg aatcattcaa gcacaaccag ataagagtga atcagagtta gtcagtcaaa 5100

taatagagca gttaataaaa aaggaaaaag tctacctggc atgggtacca gcacacaaag 5160

gaattggagg aaatgaacaa gtagatgggt tggtcagtgc tggaatcagg aaagtactat 5220

ttttagatgg aatagataag gcccaagaag aacatgagaa atatcacagt aattggagag 5280

caatggctag tgattttaac ctaccacctg tagtagcaaa agaaatagta gccagctgtg 5340

ataaatgtca gctaaaaggg gaagccatgc atggacaagt agactgtagc ccaggaatat 5400

ggcagctaga ttgtacacat ttagaaggaa aagttatctt ggtagcagtt catgtagcca 5460

gtggatatat agaagcagaa gtaattccag cagagacagg gcaagaaaca gcatacttcc 5520

tcttaaaatt agcaggaaga tggccagtaa aaacagtaca tacagacaat ggcagcaatt 5580

tcaccagtac tacagttaag gccgcctgtt ggtgggcggg gatcaagcag gaatttggca 5640

ttccctacaa tccccaaagt caaggagtaa tagaatctat gaataaagaa ttaaagaaaa 5700

ttataggaca ggtaagagat caggctgaac atcttaagac agcagtacaa atggcagtat 5760

tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag 5820

tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc 5880

aaaattttcg ggtttattac agggacagca gagatccagt ttggaaagga ccagcaaagc 5940

tcctctggaa aggtgaaggg gcagtagtaa tacaagataa tagtgacata aaagtagtgc 6000

caagaagaaa agcaaagatc atcagggatt atggaaaaca gatggcaggt gatgattgtg 6060

tggcaagtag acaggatgag gattaacaca tggaattctg caacaactgc tgtttatcca 6120

tttcagaatt gggtgtcgac atagcagaat aggcgttact cgacagagga gagcaagaaa 6180

tggagccagt agatcctaga ctagagccct ggaagcatcc aggaagtcag cctaaaactg 6240

cttgtaccaa ttgctattgt aaaaagtgtt gctttcattg ccaagtttgt ttcatgacaa 6300

aagccttagg catctcctat ggcaggaaga agcggagaca gcgacgaaga gctcatcaga 6360

acagtcagac tcatcaagct tctctatcaa agcagtaagt agtacatgta atgcaaccta 6420

taatagtagc aatagtagca ttagtagtag caataataat agcaatagtt gtgtggtcca 6480

tagtaatcat agaatatagg aaaatggccg ctgatcttca gacctggagg aggagatatg 6540

agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 6600

gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 6660

ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcctcaatg 6720

acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 6780

ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 6840

ctccaagcaa gaatcctagc tgtggaaaga tacctaaagg atcaacagct cctagggatt 6900

tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 6960

aataaatctc tggaacagat ctggaatcac acgacctgga tggagtggga cagagaaatt 7020

aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 7080

aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 7140

acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 7200

agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 7260

tcgtttcaga cccacctccc aatcccgagg ggacccgaca ggcccgaagg aatagaagaa 7320

gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc cttggcactt 7380

atctgggacg atctgcggag cctgtgcctc ttcagctacc accgcttgag agacttactc 7440

ttgattgtaa cgaggattgt ggaacttctg ggacgcaggg ggtgggaagc cctcaaatat 7500

tggtggaatc tcctacaata ttggagtcag gagctaaaga atagtgctgt tagcttgctc 7560

aatgccacag ccatagcagt agctgagggg acagataggg ttatagaagt agtacaagga 7620

gcttgtagag ctattcgcca catacctaga agaataagac agggcttgga aaggattttg 7680

ctataagctc gaaacaaccg gtacctctag aactatagct agcagatctt tttccctctg 7740

ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga 7800

aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat 7860

atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat 7920

atgccatatg ctggctgcca tgaacaaagg tggctataaa gaggtcatca gtatatgaaa 7980

cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt 8040

tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt 8100

ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc 8160

ttatgaagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc 8220

tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 8280

taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 8340

cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc 8400

atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct 8460

ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct 8520

gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta 8580

acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 8640

ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 8700

atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 8760

tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 8820

gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 8880

cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 8940

gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 9000

aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 9060

ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 9120

cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta 9180

ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 9240

cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 9300

agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 9360

gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 9420

gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 9480

tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 9540

agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 9600

agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 9660

atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 9720

cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 9780

actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 9840

aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 9900

cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 9960

ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 10020

cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 10080

ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 10140

cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 10200

ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 10260

tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 10320

ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 10380

aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 10440

gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 10500

gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 10560

ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 10620

catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 10680

atttccccga aaagtgccac ctg 10703

Claims (7)

1. a kind of CRISPR/Cas9 targeting knock outs people colon-cancer cell RITA genes and its specific sgRNA, it is characterised in that Design obtains the sgRNA of selectively targeted RITA genes Second Exon first；Secondly the sgRNA of structure RITA genes is to slowly Virus carrier system；Then this slow virus carrier system is infected into people's intestinal cancer HT-29 cells, obtained RITA Knockout cells Strain.

2. the sgRNA of selectively targeted RITA genes Second Exon according to claim 1, its DNA sequence such as SEQ Shown in ID NO.1, target sequences of the sgRNA on RITA genes is unique.

3. the sgRNA of selectively targeted RITA genes Second Exon according to claim 2, its primer include

It is located at primer pair on Second Exon for the target spot of RITA genes：

RITAsgRNAoligo1：As shown in SEQ ID NO.2；

RITAsgRNAoligo2：As shown in SEQ ID NO.3.

It is a kind of targeting knock out 4. the sgRNA of structure RITA genes according to claim 1 is to slow virus carrier system The CRISPR/Cas9 recombination expression slow virus carriers Lenti-CRISPRv2-hRITAsg of RITA genes, it is characterised in that：Contain There is the recombinant expression carrier of the sgRNA and Cas9 albumen of selectively targeted RITA genes Second Exon.

5. SEQ ID NO in the sequence of the skeleton carrier of recombinant expression carrier according to claim 4 such as sequence table：8 It is shown；

CRISPR/Cas9 targeting knock outs people colon-cancer cell RITA genes according to claim 1 and its specific SgRNA, it is characterised in that comprise the following steps：

(1) sgRNA sequences are provided, target sequences of the sgRNA on RITA genes meets 5 '-N (19) G series arrangement Rule, target sequences of the sgRNA on RITA genes are located at the extron of gene, and the sgRNA is on RITA genes Target sequence is located on the common exon of different various shear patterns, and target sequences of the sgRNA on RITA genes is only One, and target site sequences of the sgRNA on RITA be as shown in the sequence table SEQ ID NO.1 sequences, described The 5 ' of target site sequences of the sgRNA on RITA-end obtains positive oligonucleotides i.e. plus CACCG sequent synthesis Forward oligo ；The complementary strand of target site sequences of the sgRNA on RITA is obtained, and at 5 '-end of complementary strand C is added plus AAAC sequences, 3 '-end, synthesis obtains reverse oligonucleotide i.e. Reverse oligo；1 couple of synthesis is mutual Forward oligo and the Reverse oligo of the sgRNA oligonucleotides of benefit are denatured, annealed in pairs, shape after annealing Into the double-strand sgRNA oligonucleotides that can be connected into U6 carrier for expression of eukaryon；

(2) Lenti-CRISPRv2 plasmid of the sequence as shown in sequence table SEQ ID NO.8 is linearized；By the double-strand of annealing The expression vector Lenti-CRISPRv2 of carrying Cas9 gene of the sgRNA oligonucleotides with linearizing is connected acquisition carrying and contained Corresponding the sgRNA oligonucleotides of target sequence and the expression vector Lenti-CRISPRv2-hRITAsg plasmids of Cas9 genes, Transformed competence colibacillus bacterium simultaneously applies Amp+ flat boards, and picking monoclonal simultaneously goes out positive colony with universal primer U6 by sequencing identification, And bacterium, extraction plasmid are shaken to the positive colony；

（3）The expression vector for being SEQ ID NO.9 with the sequence for carrying sgRNA oligonucleotides and Cas9 genes Lenti-CRISPRv2-hRITAsg plasmids, the pLP-VSVG that sequence is SEQ ID NO.10 and SEQ ID NO.11 (AddGene 8454) and psPAX2 (AddGene 12260) packaging plasmids and package cell line, package cell line are 293TN cells.

6. pack out while carry the sgRNA and Cas9 of targeting RITA genes false type slow virus；

（4）Using the false type slow-virus infection aim cell, add after puromycin resistance screenings and further cultivate；

（5）Monoclonal cell is spread, then collects monoclonal cell, genomic DNA is extracted, using its genomic DNA as template amplification The genetic fragment of the target sequence is included, TA cloning and sequencings confirm that RITA genes have been knocked and have obtained the thin of gene knockout Born of the same parents.

7. the cell line of knockout RITA genes according to claim 1, it is people's HT-29 cells；It is characterized in that：It is HT- The cell line that RITA gene delections or insertion nucleotides in 29 cells obtain, is any in following cell line 3# and 5# Strain：Cell line 3# is：SgRNA cleavage site at 7 bases before sgRNA PAM areas on wild type RITA genes, The second exon of RITA genes is caused to lack 134 bases；Cell line 5#：Be sgRNA cleavage site be located at wild Before sgRNA PAM areas on type RITA genes at 33 bases, the second exon of RITA genes is caused to lack 160 alkali Base.